In keeping with the mission of NIDCR to improve oral, dental and craniofacial health, this proposal focuses on the developmental genetic basis of patterning of the facial skeleton. It utilizes zebrafish as a model system. The model has many positive attributes for study of the detailed nature of how embryonic cells build and shape a set of interconnected skeletal elements, and for learning about the genes that guide these cellular activities. Zebrafish broadly share basic patterning mechanisms of jaw and skull formation with other vertebrates, including humans. Furthermore, there are increasing numbers of examples known where mutations in the very same genes of humans, mouse, and zebrafish produce head skeletal malformations that are similar in detail. Hence, zebrafish can teach us much about the nature of the defects in inherited craniofacial disorders such as cleft palate. Three projects are proposed to test specific predictions of hypotheses explaining critical aspects of early skeletal patterning, and to discover new craniofacial regulatory genes. The first investigation is to examine function of a genetic pathway that is activated in the embryonic pharyngeal arches by an upstream signaling molecule, Endothelin1, critical for jaw patterning in zebrafish and mouse alike. With even mild disruption of this pathway the joint between the upper and lower jaw is lost, replaced with cartilage in a way that seems to mimic early stages of severe osteoarthritis. The experiments examine specific roles of Endothelin1 target genes that normally regulate repression of cartilage development where the joint arises. All of the zebrafish craniofacial studies carried out so far have been limited to the first few days of skeletal development, and the second and third projects will expand our knowledge to include many more days, to the juvenile-adult stage. The second project utilizes confocal time-lapse recording of development of fluorescently labeled bone and bone-forming cells in living, intact fish larvae. It will explore the nature of the dynamic cellular reorganizations that appear to underlie formation of new bone morphologies. The third investigation is to use a skeletal mutant screen at the juvenile-adult stage to discover new craniofacial genes. The highest priority is to find genes that function to expand upon the early skeletal scaffold that forms in the embryo. Analyses of the mutants recovered in this screen will reveal how the genes work.